Novel complex of human kallikrein 2 (hK2) and protease inhibitor-6 (PI-6) in prostate tumor tissue and methods of using the complex

ABSTRACT

The present invention provides a novel complex of hK2 and PI-6 and methods of using the novel complex. The novel complexes of hK2 and PI-6 of the present invention exist at an elevated level in prostate cancer tissues. Therefore, the hK2-PI6 complexes of the present invention may be used as a serum marker for detecting prostate cancer. They may also be used as an immunohistological marker to detect prostate cancer tissues. In accordance with the present invention, the hK2-PI6 complexes of the present invention may be detected in patient tissue samples by immunohistochemical and/or in patient fluid samples by in vitro immunoassay procedures. Diagnostic kits and diagnostic methods for detecting the existence of prostate cancer are also provided.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to the prostate and specificallyto a novel complex of hK2 and PI-6 formed in the prostate, and methodsof using the novel complex.

[0003] 2. Description of the Prior Art

[0004] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of this application, preceding the claims.

[0005] Three members of the human kallikrein family have been identifiedso far, designated hK1, hK2 and hK3 (1). All are serine proteases withhigh sequence identity. Two of these kallikreins, hK2 and hK3, are foundalmost exclusively in the prostate (extensively reviewed in (2). hK3,known more commonly as prostate-specific antigen, PSA, is a widely usedserum marker for prostate. More recently, hK2 has become the focus ofinvestigations into its possible role as a prostate cancer marker, aswell as possible roles in prostate cancer biology (2).

[0006] hK2 is similar to PSA in many respects such as prostate tissuelocalization (3; 4), 80% sequence identity (5; 6), and regulation byandrogens (7; 8). From a biochemical perspective, hK2 is different fromPSA in that it shows a strong trypsin-like activity while PSA has weakchymotrypsin-like activity. However, unlike true kallikreins, hK2 showslittle or no kininogenase-like activity (9; 10) and so does not appearto function primarily as a prostatically-expressed kininogenase.

[0007] The physiological roles for hK2 have not been established, thoughseveral activities have been described. hK2 has been shown to activatethe zymogen form of PSA (pPSA) (11-13), and the zymogen of hK2(autoactivation) (14). In this respect hK2 is unique from the otherhuman tissue kallikreins, hK1 and PSA. The activation of pPSA by hK2 isparticularly interesting since it suggests a possible physiological rolefor hK2 in the regulation of PSA activity.

[0008] Immunohistochemical studies using hK2-specific monoclonalantibodies have shown hK2 to be more highly expressed in prostatecarcinoma than in normal tissues (15). This is the inverse of PSA whichtends to be lower in more poorly differentiated cancer epithelium thanin normal tissues.

[0009] hK2 has been shown to activate urokinase-type plasminogenactivator (uPA) by cleavage of the single-chain uPA at Lys¹⁵⁸ togenerate the two-chain active form of uPA (12; 16). It has been recentlyreported that hK2 rapidly complexes with PAI-1 in vitro and that hK2also inactivates about 6 moles of PAI-1 during complex formation (17).It is therefore possible that the elevated levels of hK2 in prostatecancer play a biological role either by the activation of urokinase orby the inactivation of PAI-1, the primary inhibitor of urokinase.

[0010] Physiologically hK2 has been identified as a complex with ACT inserum (18)and PCI in seminal plasma (19). However, it was not previouslyknown whether hK2 forms any tissue specific complex in the prostate. Thephysiological and biological role of hK2 in the prostate was also notknown. Therefore, a need exists to study the role of hK2 in theprostate, particularly in prostate cancer.

SUMMARY OF THE INVENTION

[0011] The present invention is based on the discovery of a complex inprostate tissue extracts consisting of hK2 and a serine proteaseinhibitor known as protease inhibitor-6 (PI-6). PI-6 was first reportedin placental tissue where it was called placental thrombin inhibitor dueto its ability to form in vitro complex with thrombin (20). It has alsobeen called CAP, cytoplasmic antiprotease, since it has been shown to becytoplasmically localized (21). PI-6 is expressed in epithelial andendothelial cells and has been described in a number of human tissuesand cells including kidney, heart, skeletal muscle and platelets(22-24). It has not been reported in the prostate. In all cases so farPI-6 appears to be cytoplasmically localized.

[0012] The discovery of hK2-PI6 complex in the prostate is unique fromthe previous reports of hK2-ACT and hK2-PCI in at least two majorrespects: 1) PI-6 itself is a novel and relatively newly describedmember of the serine protease inhibitor family which has not beenreported in prostate tissues; and 2) PI-6 is thought to beintracellular, which suggests either a novel pathway for the formationwith the putatively extracellular hK2, or an altered pathway, possiblydue to the oncogenic process.

[0013] While not wanting to be bound by the theory, it is believed thatthe finding of prostatic PI-6 in complex with hK2 suggests that PI-6 mayplay certain roles in the prostate. PI-6 represents a tissue-specificinhibitor of hK2 which may have biological significant in the regulationof hK2, and may play a role in prostate cancer biology.

[0014] It is a discovery of the present invention that the hK2-PI6complex is elevated in prostate tumor compared to benign or normalprostate tissue. It is also a discovery of the present invention thatthe level of PI-6 is elevated in prostate tumor. Therefore, both thecomplex and the level of hK2 or PI-6 alone may be a novel marker forprostate cancer.

[0015] Accordingly, one aspect of the present invention provides anisolated and substantially pure complex consisting of proteaseinhibitor-6 (PI-6) and human kallikrein 2 (hK2). The hK2 contained inthe complex may be an intact hK2 or a fragment of hK2. The complex ofthe present invention exists at an elevated level in a prostate tumortissue.

[0016] Another aspect of the present invention provides an antibody thatis specifically immunoreactive with a complex consisting of proteaseinhibitor-6 (PI-6) and human kallikirein 2 (hK2). The antibody may be apolyclonal antibody or a monoclonal antibody.

[0017] A further aspect of the present invention provides a method ofdetecting or determining in a sample a complex consisting of proteaseinhibitor-6 (PI-6) and human kallikrein 2 (hK2). The method comprisesthe steps of:

[0018] (a) contacting an amount of an agent which specifically binds tothe complex to be detected with the sample under a condition that allowsthe formation of a ternary complex comprising the agent and the hK2-PI6complex; and

[0019] (b) detecting or determining the presence or amount of theternary complex.

[0020] In preferred embodiments, the agent comprises an antibody. Thesample to be detected may be a mammalian tissue sample, particularly aprostate tissue sample, or a sample of human physiological fluidincluding, but not limited, to serum, seminal plasma, urine, and blood.

[0021] Yet another aspect of the present invention provides animmunoassay for detecting hK2-PI6 complexes in a biological fluidsample. The immunoassay comprises the steps of:

[0022] (a) providing a first antibody that recognizes one epitope of thehK2-PI6 complex, and a second antibody that recognizes another epitopeof the hK2-PI6 complex,

[0023] (b) contacting the first antibody with the sample under acondition that allows the first antibody to bind to the complex to forma first complex that comprises the first antibody and the hK2-PI6complex,

[0024] (c) contacting the second antibody with the first complex under acondition that allows the second antibody to bind to the hK2-PI6 complexto form a second complex that comprises the first antibody, the secondantibody and the hK2-PI6 complex, and

[0025] (d) detecting and determining the second complex as a measurementof the hK2 PI-6 complex contained in the sample.

[0026] According to embodiments of the present invention, the first andsecond antibodies are respectively an antibody that is specificallyimmunoreactive for the hK2-PI6 complex. Alternatively, one of the firstantibody and second antibodies is an antibody against hK2, and anotherof the first and second antibodies is an antibody against PI 6.

[0027] A further aspect of the present invention provides a diagnostickit for detecting or determining in a sample a complex consisting ofPI-6 and hK2. The kit comprises a known amount of an agent whichspecifically binds to the complex, wherein the agent is detectablylabeled or binds to a detectable label.

[0028] According to embodiments of the present invention, the sample isa sample of human physiological fluid or a human prostate tissue sampleand the agent comprises an antibody that specifically binds to thecomplex.

[0029] The present invention also provides a diagnostic kit fordetecting or determining in a sample a complex consisting of PI-6 andhK2. The kit comprises a known amount of a first agent whichspecifically binds to the complex, wherein the first agent is detectablylabeled or binds to a detectable label, and a known amount of a secondagent which specifically binds to the complex, wherein the second agentis bound to a solid support.

[0030] According to embodiments of the present invention, the firstagent comprises an antibody that is specifically immunoreactive with thecomplex, and the second agent comprises an antibody that is specificallyimmunoreactive with the complex. Alternatively, one of the first andsecond agents comprises an antibody that is specifically immunoreactivewith hK2, and another of the first and second agents comprises anantibody that is specifically immunoreactive with PI-6.

[0031] The present invention further provides a diagnostic method fordetermining the presence or absence of prostate cancer comprising:

[0032] (a) contacting an amount of an agent, which specifically binds toa complex consisting of PI-6 and hK2, with a sample obtained from ahuman containing the complex under a condition sufficient to allow theformation of a ternary complex comprising the agent, the PI-6, and thehK2; and

[0033] (b) determining the amount of the ternary complex in the sampleand correlating the amount of the ternary complex to the presence orabsence of prostate cancer in the human.

[0034] According to embodiments of the present invention, the sample isa sample of physiological fluid or a prostate tissue sample. The agentcomprises an antibody.

[0035] The present invention further provides a diagnostic method fordetermining the presence or absence of prostate cancer. The methodcomprises:

[0036] (a) contacting an amount of an agent, which specifically binds toPI-6, with a sample obtained from a human containing the PI-6 under acondition sufficient to allow the formation of a binary complexcomprising the agent and the PI-6, wherein the sample is selected from agroup consisting of a prostate tissue sample, serum, seminal plasma,urine and blood; and

[0037] (b) determining the amount of the complex in the sample andcorrelating the amount of the complex to the presence or absence ofprostate cancer in the human.

[0038] According to embodiments of the present invention, the sample isa prostate tissue sample, and the agent comprises an antibody.

[0039] The invention is defined in its fullest scope in the appendedclaims and is described below in its preferred embodiments.

DESCRIPTION OF THE FIGURES

[0040] The above-mentioned and other features of this invention and themanner of obtaining them will become more apparent, and will be bestunderstood by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict only atypical embodiment of the invention and do not therefore limit itsscope. They serve to add specificity and detail, in which:

[0041]FIG. 1 shows a Western blot of prostate tissue extracts. Thisfigure shows an example of higher hK2-PI6 complex in tumor tissue thanbenign.

[0042]FIG. 2 shows a Western blot of hK2-complex standards and prostatetissue extract.

[0043]FIG. 3 shows N-terminal sequence of a blot of purified hK2 formsfrom prostate tissues.

[0044]FIG. 4 is the reversed phase HPLC profile of the peptidesgenerated after trypsin digestion of the 50 kDa hK2-PI6 band excisedfrom a gel.

[0045]FIG. 5 is a Western blot of hK2-PI6 complex formed in vitro, asdetected with the rabbit anti-PI-6 polyclonal antibody of the presentinvention and hK2 antibody.

[0046]FIG. 6 shows a Western blot of prostate tissue extracts probedwith the rabbit anti-PI-6 polyclonal antibody of the present invention.

[0047]FIG. 7 shows the hK2-PI6 complex in prostate tissue extracts asdetected by the rabbit polyclonal anti-PI-6 antibody of the presentinvention, and also as detected with anti-hK2 antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0048] One aspect of the present invention provides an isolated andsubstantially pure complex consisting of protease inhibitor-6 (PI-6) andhuman kallikrein 2 (hK2). The term “substantially pure,” as used herein,refers to a complex, which consists of protease inhibitor-6 (PI-6) andhuman kallikrein 2 (hK2), that is substantially free of other proteins,lipids carbohydrates or other materials with which it is naturallyassociated. The substantially pure hK2-PI6 complex of the presentinvention will yield a single major band on a non-reducingpolyacrylamide gel. The complex has a molecular weight of about 64 kDameasured by SDS-PAGE technique. The purity of the specific form of hK2can also be determined by amino-terminal amino acid sequence analysis.

[0049] For the purpose of the present invention, the term hK2 mayinclude an intact hK2 polypeptide and a fragment of hK2 polypeptide. Forexample, in one embodiment of the present invention, the hK2 polypeptidecontained in an hK2-PI6 complex of the present invention may be clippedat arginine 145 of the hK2 polypeptide sequence. Therefore, when anhK2-PI6 complex with the clipped hK2 is purified through a reducingpolyacrylamide gel, the complex will contain PI-6 and a fragment of hK2that is covalently attached to PI-6. Another clipped fragment which isnot covalently attached to PI-6 will be separated from the complex underreducing and denaturing conditions. For example, in one embodiment, anhK2-PI6 complex may consist of PI-6 and the hK2 145-237 fragment. Such acomplex has a molecular weight of about 50 kDa measured by a reducingpolyacrylamide gel technique. It should be understood that the hK2contained in the complex of the present invention may be clipped atother locations of the hK2 sequence. Therefore, for the purpose of thepresent invention, the term hK2 as used for an hK2-PI6 complex mayinclude any hK2 fragment clipped at any position of intact hK2 as longas the clipped fragment is covalently attached to the PI-6 of thecomplex.

[0050] It is a discovery of the present invention that PI-6 exists inthe prostate tumor. It is also a discovery of the present invention thatthe hK2-PI6 complex exists in prostate tumor at an elevated level. Thelevel of the hK2-PI6 complex is elevated if the amount of the complex ishigher than the amount of the complex that exists in normal or benigntissues.

[0051] The hK2-PI6 complex of the present invention may be isolated froma prostate tissue by methods described herein, i.e., by in vitropreparation pure or semi-purified proteins, and also by any othermethods known to one skilled in the art for protein purifications. Thepurified hK2-PI6 complex may be used for antibody development.Accordingly, one aspect of the present invention provides an antibodythat is specifically immunoreactive with and binds to an hK2-PI6 complexof the present invention. The term “specifically immunoreactive” or“specific” as used herein indicates that the antibodies of the presentinvention only recognize and bind to hK2-PI6 complexes of the presentinvention, not the free hK2 or free PI-6.

[0052] An antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations, are provided. Monoclonal antibodiesare made from an antigen containing a complex of the present inventionor a portion of the complex of the present invention by methods wellknown in the art (E. Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988). In general, this method involvespreparing an antibody-producing fused cell line, e.g., from primaryspleen cells fused with a compatible continuous line of myeloma cells,growing the fused cells either in mass culture or in an animal speciesfrom which the myeloma cell line used was derived or is compatible. Suchantibodies offer many advantages in comparison to those produced byinoculation of animals, as they are highly specific and sensitive andrelatively “pure” immunochemically. Immunologically active fragments ofantibodies are also within the scope of the present invention, e.g., thef(ab) fragment, as are partially humanized monoclonal antibodies.

[0053] If desired, polyclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which a polypeptideor a peptide to which the antibodies were raised is bound. Those ofskill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. (See, for example, Coliganet al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991, incorporated by reference.)

[0054] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, F(ab′)₂ and Fv,which are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with its antigen orreceptor and are defined as follows:

[0055] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of thewhole antibody with the enzyme papain to yield an intact light chain anda portion of one heavy chain;

[0056] (2) Fab′, the fragment of an antibody molecule, can be obtainedby treating the whole antibody with pepsin, followed by reduction, toyield an intact light chain and a portion of the heavy chain—two Fab′fragments are obtained per antibody molecule;

[0057] (3) F(ab′)₂, the fragment of the antibody that can be obtained bytreating the whole antibody with the enzyme pepsin without subsequentreduction—F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

[0058] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0059] (5) Single chain antibody (“SCA”), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

[0060] Methods of making these fragments are known in the art. (See, forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference.)

[0061] As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules, such as amino acids or sugar side chains, andusually have specific three-dimensional structural characteristics, aswell as specific charge characteristics.

[0062] Since hK2-PI6 complexes of the present invention are elevated inprostate cancer tissues, they may be used as a serum marker fordetecting prostate cancer. They may also be used as animmunohistological marker to detect prostate cancer tissues. Inaccordance with the present invention, the hK2-PI6 complexes of thepresent invention may be detected in patient tissue samples byimmunohistochemical and/or in patient fluid samples by in vitroimmunoassay procedures. The determination of the complexes of thepresent invention in a patient sample is of significant diagnosticutility and may be an indicator of or correlate with the progression ofa prostate cancer patient.

[0063] Immunohistochemical methods for the detection of antigens inpatient tissue specimens are well known in the art and need not bedescribed in detail herein. For example, methods for theimmunohistochemical detection of antigens are generally described inTaylor, Arch. Pathol.Lab. Med. 102:113 (1978). Briefly, in the contextof the present invention, a tissue specimen obtained from a patientsuspected of having a prostate related problem is contacted with anantibody, preferably a monoclonal antibody, recognizing a complex of thepresent invention. The site at which the antibody is bound is thereafterdetermined by selective staining of the tissue specimen by standardimmunohistochemical procedures.

[0064] In an alternative embodiment, antibodies that are specific foreither hK2 or PI-6 are used for detecting and determining the hK2-PI6complexes of the present invention in a tissue specimen. According tothis embodiment, the immunohistochemical procedure includes the stepsof:

[0065] (a) providing a first antibody that is specific for free hK2, asecond antibody that is specific for PI-6, and a third antibody thatrecognizes and binds a hK2-PI6 complex of the present invention;

[0066] (b) contacting the first and second antibodies with the tissuespecimen under a condition that all the free hk2 and free PI-6 areblocked by the first and the second antibodies respectively;

[0067] (c) contacting the third antibody with the tissue specimen undera condition that allows the third antibody to bind the hK2-PI6 complexcontained in the tissue specimen to form a ternary complex comprisingthe antibody and the hK2-PI6 complex; and

[0068] (d) detecting or determining the presence or amount of theternary complex.

[0069] In accordance with embodiments of the present invention, thethird antibody may be an antibody against hK2, which antibody recognizesand binds not only free hK2 but also the hK2-PI6 complex of the presentinvention. Alternatively, the third antibody may be an antibody againstPI-6, which antibody recognizes and binds to not only free PI-6, butalso the hK2-PI6 complex.

[0070] It should be understood that if the hK2-PI6 complex is a dominanthK2 complex in a sample, such as a prostate tissue sample, onlyantibodies that recognize free hK2 and antibodies that recognize bothfree hk2 and hK2 complexes may be used for the purpose of detecting thehk2-PI-6 complex. In this case, it may not be necessary for the use ofantibodies against PI-6.

[0071] Antibodies against hK2 or PI-6 may be generated by the methodsdescribed herein and by methods that are commonly known in the art,using hK2 or PI-6 as antigens. Antibodies that recognize only free hK2or PI-6 are those that recognize epitopes that are blocked by theformation of hK2-PI6 complex. Antibodies that recognize both free hK2 orfree PI-6 and a hK2-PI6 complex are those that recognize epitopes thatare not blocked by the formation of hK2-PI6 complex. The characteristicof hK2 or PI-6 antibodies may be determined by Western blot or any othermethods known to one skilled in the art.

[0072] In one embodiment of the present invention, the tissue specimenis a tissue specimen obtained from the prostate of a patient. Theprostate tissue may be a normal prostate tissue, a cancer prostatetissue or a benign prostatic hyperplasia tissue.

[0073] The general methods of the in vitro detection of antigenicsubstances in patient fluid samples by immunoassay procedures are alsowell known in the art and require no repetition herein. For example,immunoassay procedures are generally described in Paterson et al., Int.J. Can. 37:659 (1986) and Burchell et al., Int. J Can. 34:763 (1984).According to one embodiment of the present invention, an immunoassay fordetecting the complex of the present invention in a biological samplecomprises the steps of (a) contacting an amount of an agent whichspecifically binds to an hK2-PI6 complex to be detected with the sampleunder a condition that allows the formation of a ternary complexcomprising the agent and the hK2-PI6 complex, and (b) detecting ordetermining the presence or amount of the ternary complex as ameasurement of the amount of the hK2-PI6 complex contained in thesample.

[0074] For the purpose of the present invention, the biological samplecan be any human physiological fluid sample that contains hK2-PI6complexes of the present invention. Examples of the human physiologicalfluid sample include, but are not limited to, serum, seminal plasma,urine and blood.

[0075] For the purpose of the present invention, both monoclonalantibodies and polyclonal antibodies may be used as long as suchantibodies possess the requisite specificity for the antigen provided bythe present invention. Preferably, monoclonal antibodies are used.

[0076] Monoclonal antibodies can be utilized in liquid phase or bound toa solid phase carrier. Monoclonal antibodies can be bound to manydifferent carriers and used to determine hK2-PI6 complexes of thepresent invention. Examples of well-known carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, agaroses andmagnetites. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Examples of insoluble carriers include,but are not limited to, a bead and a microtiter plate. Those skilled inthe art will know of other suitable carriers for binding monoclonalantibodies, or will be able to ascertain such by routineexperimentation.

[0077] In addition, the monoclonal antibodies in these immunoassays canbe detectably labeled in various ways. For example, monoclonalantibodies of the present invention can be coupled to low molecularweight haptens. These haptens can then be specifically detected by meansof a second reaction. For example, it is common to use such haptens asbiotin, which reacts with avidin, or dinitrophenyl, pyridoxal andfluorescein, which can react with specific anti-hapten antibodies. Inaddition, monoclonal antibodies of the present invention can also becoupled with a detectable label such as an enzyme, radioactive isotope,fluorescent compound or metal, chemiluminescent compound orbioluminescent compound. Furthermore, the binding of these labels to thedesired molecule can be done using standard techniques common to thoseof ordinary skill in the art.

[0078] One of the ways in which the antibodies can be detectably labeledis by linking it to an enzyme. This enzyme, in turn, when later exposedto its substrate will react with the substrate in such a manner as toproduce a chemical moiety which can be detected by, for example,spectrophotometric or fluorometric means (ELISA system). Examples ofenzymes that can be used as detectable labels are horseradishperoxidase, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

[0079] For increased sensitivity in the ELISA system, the proceduresdescribed can be modified using biotinylated antibodies reacting withavidin-peroxidase conjugates.

[0080] The amount of antigen can also be determined by labeling theantibody with a radioactive isotope. The presence of the radioactiveisotope would then be determined by such means as the use of a gammacounter or a scintillation counter. Isotopes which are particularlyuseful are ³H, ¹²⁵I, ¹²³I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe,⁷⁵Se, ¹¹¹n, ⁹⁹mTc, ⁶⁷Ga, and ⁹⁰Y.

[0081] Determination of the antigen is also possible by labeling theantibody with a fluorescent compound. When the fluorescently labeledmolecule is exposed to light of the proper wave length, its presence canthen be detected due to fluorescence of the dye. Among the mostimportant fluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde,and fluorescamine.

[0082] Fluorescence emitting metal atoms such as Eu (europium), andother lanthanides, can also be used. These can be attached to thedesired molecule by means of metal-chelating groups, such as DTPA orEDTA.

[0083] Another way in which the antibody can be detectably labeled is bycoupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged immunoglobulin is then determined by detectingthe presence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, aromatic acridinium ester, imidazole,acridinium salt, and oxalate ester.

[0084] Likewise, a bioluminescent compound may also be used as a label.Bioluminescence is a special type of chemiluminescence which is found inbiological systems and in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent molecule would be determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase, and aequorin.

[0085] Qualitative and/or quantitative determinations of hK2-PI6complexes of the present invention in a sample may be accomplished bycompetitive or non-competitive immunoassay procedures in either a director indirect format. Examples of such immunoassays are theradioimmunoassay (RIA) and the sandwich (immunometric) assay. Detectionof the antigens using the monoclonal antibodies of the present inventioncan be done utilizing immunoassays which are run in either the forward,reverse, or simultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

[0086] The term “immunometric assay” or “sandwich immunoassay” includesa simultaneous sandwich, forward sandwich and reverse sandwichimmunoassay. These terms are well understood by those skilled in theart. Those of skill will also appreciate that antibodies according tothe present invention will be useful in other variations and forms ofassays which are presently known or which may be developed in thefuture. These are intended to be included within the scope of thepresent invention.

[0087] In accordance with one embodiment of the present invention,antibodies that are specific for free hK2 or PI-6 may also be used in animmunoassay of the present invention for detecting or determining in asample of a human physiological fluid the hK2-PI6 complex of the presentinvention. For example, antibodies against hK2 or PI-6 may be used in asandwich assay of the present invention. Accordingly, one embodiment ofthe present invention provides an immunoassay for detecting hK2-PI6complexes in a sample, the assay including the steps of:

[0088] (a) providing a first antibody that recognizes one epitope of thehK2-PI6 complex, and a second antibody that recognizes another epitopeof the hK2-PI6 complex,

[0089] (b) contacting the first antibody with the sample under acondition that allows the first antibody to bind to the complex to forma first complex that comprises the first antibody and the hK2-PI6complex,

[0090] (c) contacting the second antibody with the first complex under acondition that allows the second antibody to bind to the hK2-PI6 complexto form a second complex that comprises the first antibody, the secondantibody and the hK2-PI6 complex, and

[0091] (d) detecting and determining the second complex as a measurementof the hK2-PI6 complex contained in the sample.

[0092] The first and second antibodies may be antibodies that arespecifically immunoreactive with hK2-PI6 complexes of the presentinvention. Alternatively, the first antibody may be an antibody that isspecific for hK2, and the second antibody may be an antibody that isspecific for PI-6, or vise versa. Preferably, one of the first andsecond antibodies is bound to a solid support whereas another antibodyis labeled with a detecting or detectable label by methods describedherein. One skilled in the art should be able to design a propersandwich assay without undue experimentation by using antibodiesspecific for hK2-PI6 complexes of the present invention or antibodiesagainst hK2 and PI-6 in view of the teachings of the present invention.

[0093] One aspect of the present invention provides a diagnostic kit fordetecting or determining in a sample a complex consisting of PI-6 andhK2. The kit comprises a known amount of an agent which specificallybinds to the complex, wherein the agent is detectably labeled or bindsto a detectable label. For the purpose of the present invention, thesample may be a sample of human physiological fluid such as, but notlimited to, serum, seminal plasma, urine and blood. The sample may alsobe a tissue specimen coming from the prostate of a patient. The agentmay be an antibody that specifically binds to hK2-PI6 complexes of thepresent invention. Preferably the agent is a monoclonal antibody,although a polyclonal antibody may also be used.

[0094] In another embodiment of the present invention, a diagnostic kitmay comprise two known amounts of agents, wherein one agent recognizesone epitope of the hK2-PI6 complex, and another agent recognizes adifferent epitope of the hK2-PI6 complex. Preferably, one agent islabeled with a detectable label, and the another agent is bound to asolid support. In one embodiment of the present invention, both agentscomprise respectively an antibody that is specifically immunoreactive toan hK2-PI6 complex of the present invention. In another embodiment ofthe present invention, one agent comprises an antibody that is specificfor hK2, and the another agent comprises an antibody that is specificfor PI-6.

[0095] Another aspect of the present invention provides a diagnosticmethod for determining the presence or absence of prostate cancer. Themethod includes the steps of:

[0096] (a) contacting an amount of an agent, which specifically binds toa complex consisting of PI-6 and hK2, with a sample obtained from ahuman containing the complex under a condition sufficient to allow theformation of a ternary complex comprising the agent, the PI-6, and thehK2, and

[0097] (b) determining the amount of the ternary complex in the sampleand correlating the amount of the ternary complex to the presence orabsence of prostate cancer in the human.

[0098] In one embodiment of the present invention, the sample may be asample of human physiological fluid including, but not limited to,serum, seminal plasma, urine, and blood. In another embodiment of thepresent invention, the sample may be tissue specimen from the prostateof a patient. For the purpose of the present invention, the agent may bean antibody that specifically recognizes hK2-PI6 complexes of thepresent invention. The antibody may be a monoclonal antibody or apolyclonal antibody.

[0099] It is a discovery of the present invention that PI-6 exists inprostate cancer tissues. It is also a discovery of the present inventionthat PI-6 exists at an elevated level as a complex with hK2 in prostatecancer. It is thus possible that PI-6 or some specific form of complexedPI-6 may show a correlation with prostate cancer. Therefore, thedetection and measurement of PI-6 or a specific form of complexed PI-6may be important in determining the existence of cancer in prostate orother tissue samples. For example, PI-6 may be used as animmunohistological marker to detect prostate cancer tissues. PI-6 of thepresent invention may be detected in patient tissue samples byimmunohistochemical procedures that are described herein.

[0100] Accordingly, one aspect of the present invention provides adiagnostic method for determining the presence or absence of prostatecancer, the method comprising the steps of:

[0101] (a) contacting an amount of an agent, which specifically binds toPI-6, with a sample obtained from a human containing the PI-6 under acondition that allows the formation of a binary complex comprising theagent and the PI-6; and

[0102] (b) determining the amount of the complex in the sample andcorrelating the amount of the complex to the presence or absence ofprostate cancer in the human.

[0103] For the purpose of the present invention, the sample may be asample from a prostate or other tissue. In one embodiment, the agentcomprises an antibody that is specific for PI-6.

[0104] The following examples are intended to illustrate, but not tolimit, the scope of the invention. While such examples are typical ofthose that might be used, other procedures known to those skilled in theart may alternatively be utilized. Indeed, those of ordinary skill inthe art can readily envision and produce further embodiments, based onthe teachings herein, without undue experimentation.

EXAMPLE I Characterization of hK2-PI6 Complexes in Prostate TissueMaterial and Methods

[0105] Material

[0106] PAI-1 was obtained from Oncogene Research Products (San Diego,Calif.). ACT was obtained from Athens Research (Athens, Ga.). PurifiedRecombinant hK2 were expressed in the syrian hamster carcinoma cellline, AV12, and immunoaffinity purified using the murine hK2-specificmonoclonal antibody HK1G586.1 as described previously (14; 25).HK1G586.1 has been shown to have negligible cross-reactivity with PSA(18; 26). PF1D215 is a monoclonal developed toward PSA fragments, butwhich is also cross-reactive with hK2. PSM 773 is a mAb which specificfor PSA and does not cross react with hK2. The above antibodies weredeveloped at Hybritech (San Diego, Calif.).

[0107] Methods

[0108] Extraction and SDS PAGE of Prostate Tissues

[0109] Prostatectomy tissues were frozen in liquid nitrogen, pulverized,then homogenized in PBS buffer containing a cocktail of proteaseinhibitors (Complete, Boeringer Manheim). SDS PAGE was performed on aNovex mini-gel with 4-20% gradient. Each lane was loaded with theextraction supernatant solution which contained 5 ug of total protein.Samples were electroblotted onto nitrocellulose for Western blotanalysis and PVDF for N-terminal sequencing. Primary antibodiesHK1G586.1 and PF1D215.2 were used at 5 ug/ml and secondary antibody(Goat anti-mouse-HRP, 1:50,000, Jackson Immunoresearch Laboratories,Inc., West Grove, Pa.) was used to probe the blots. The immunoreactivesignals were detected by ECL Ultra (Amersham, Buckinghamshire, England)according to manufacturer's instructions.

[0110] Preparation of hK2-complex Standards

[0111] In vitro complexes of hK2 with ACT, PCI and PAI-1 were preparedby incubation of hK2 with excess inhibitor as described previously (17).

[0112] N-terminal Sequence Analysis

[0113] N-terminal analysis was performed on a PE-Applied BiosystemsModel 492 amino acid sequencer. Protein bands blotted onto PVDF werevisualized by Coomassie Brilliant Blue R-250, excised and applied to thesequencer.

[0114] Samples for internal sequencing were first reduced with 2 mm DTTand alkylated with iodoacetamide prior to SDS-PAGE. Coomassie-stainedbands from the gel were excised, cut into small pieces, washed 2× with50% acetonitrile/0,2M sodium bicarbonate for 30 min, and completelydried in a speedvac. Dried gel slices were then reconstituted with 50 ul0,2M sodium bicarbonate containing 1 ug of sequencing grade trypsin(Promega). Samples were digested for 20 h at 37° C.

Results

[0115] Benign and cancerous prostate tissues were homogenized andextracted. The supernatant solutions were subjected to non-reducingSDS-PAGE and analyzed by Western blot (FIG. 1). FIG. 1, lanes 1 and 2were probed with the hK2-specific monoclonal antibody, hK1G586.1, andshows the free and complexed hK2 present in benign prostate tissue andtumor, respectively. The band at 33K is hK2 and the band at 64Kindicates a complex of hK2. The level of complexed hK2 is higher in thetumor extract. The mobility of this complex indicates a molecular massof 64 kDa, lower than the known physiological hK2 complexes of ACT andPCI (see FIG. 2).

[0116] Lanes 3 and 4 were probed with PSM773 to detect PSA and PSAcomplexes. No detectable higher molecular mass PSA complex was observedeven after extended exposure times. Immunoassay measurements of the hK2and PSA in these extracts gave a similar value for benign tissue andtumor. Total PSA was approximately 10 ug per mg protein and total hK2was 0.3 ug per mg protein. 5 ug of total protein was loaded in eachlane. Thus, significant levels of hK2 are found as a complex in prostatetissue, and this complex is elevated in prostate tumor. In contrast, themuch higher levels of PSA are predominantly in the free, uncomplexedform. FIG. 1 further indicates that the 64 kDa hK2-complex is the majorkallikrein complex found in prostate tissue extracts.

[0117] In order to obtain enough material to identify the inhibitor inthe hK2-complex, approximately 50 g of prostate tissue was extracted.The hK2 and hK2-complex were purified from the extract by passage overan immunoaffinity column containing the hK2 specific monoclonalantibody, HK1 G586.1. FIG. 2 shows the Western blot profile of thepurified hK2 forms eluted from the affinity column together withhK2-complex standards prepared in vitro. Lane 1 shows purifiedrecombinant hK2 and lanes 2, 3 and 4 contain hK2 complexes with ACT, PCIand PAI-1, respectively. The mobilities of these hK2 complexes aredifferent due to the differences in the molecular mass of theinhibitors. For example, lane 2 is hK2 incubated with ACT which forms a90 kDa complex; lane 3 is hK2 incubated with PCI which forms a 75 kDacomplex; lane 4 is hK2 incubated with PAI-1 which forms a 66 kDacomplex.

[0118] Lane 5 shows the purified hK2 forms extracted from the tissue.When probed with HK1G586.1 a faint band at 64 kDa is detected, togetherwith strong bands at 33 kDa and 22 kDa. The 22 kDa is commonly presentin hK2 preparations and can be seen as faint bands in the standard hK2samples in lanes 1-4. This band is a fragment of hK2 which results froma cleavage at arginine 145. The epitope for HK1G586.1 is thus shown tobe on the N-terminal side of this clip.

[0119] Lane 6 is the same as lane 5 except that this lane was probedwith PF1D215. PF1D215 was developed towards fragments of PSA but iscross-reactive with hK2. Lane 6 shows a different banding pattern thanlane 4. Both the 30 kDa and the faint 64 kDa band are detected, but 2new major bands at 10 kDa and 50 kDa are detected. A faint bandmigrating at the same position as hK2-ACT is also detected.

[0120] The identity of the bands in FIG. 2 were determined by N-terminalsequencing a blot of the same sample. In FIG. 3, 15 uL of theconcentrated, purified tissue hK2 containing approximately 10 ug of freehK2 was loaded in this lane. Each band on PVDF was excised and subjectedto N-terminal sequencing through nine cycles. Bands 1 and 2 are hK2fragments of hK2. Band 1 begins with the sequence, SLQXVSLHL whichcorresponds to the hK2 sequence beginning at serine 146. Together withthe molecular mass of 10 kDa, this indicates that this fragment is hK2146-237. Band 2 begins with the N-terminal sequence for hK2 and isconsistent with the hK2 fragment from residues 1-145. Band 3, at 33 kDa,also begins with the N-terminal sequence which is consistent with intacthK2.

[0121] Band 4, the 50 kDa band seen in FIG. 2, lane 6, begins with thesequence SLQXVSLHL. This is the same sequence as band 1, suggesting thatthis is the 10 kDa fragment of hK2 covalently attached to the inhibitor.However, the absence of a second sequence indicated that that N-terminusof the inhibitor was blocked. The reactive serine in hK2 which forms thecovalent bond with serpins is residue 189, which is C-terminal to the145 clip. This is consistent with FIG. 2, lane 6, where PF1D215.2 wasshown to recognize the 10 kDa fragment of hK2 146-237, and to detect the50 kDa complex consisting of the hK2 146-237 covalently attached to theinhibitor.

[0122] Band 5, corresponding to the faint 64 kDa band in FIG. 2,contained the hK2 N-terminus sequence. Since this molecular mass ishigher than intact hK2, it is likely that this band contains intact hK2attached to a blocked inhibitor. The stronger band just above band 5 wasdetermined to be human serum albumin by sequencing. And finally, band 6had two sequences: intact hK2 and ACT, which indicates that this isintact hK2 bound to ACT.

[0123] The sequences in FIG. 3 indicate that the vast majority of thehK2 is clipped at arginine 145 and that the majority of the hK2 bound tothe blocked inhibitor (at 50 kDa) is also clipped. The relativepercentage of hK2 in each band was calculated, based on the total pmolof all bands measured during sequence analysis. Band 1 contained 48pmoles of the 10 kDa hK2 fragment which represented 73% of the total hK2sequenced on this blot. Band 2, the 22 kDa fragment, contained 84% or11% higher hK2 than band 1. The additional 22 kDa hK2 fragment waspresumably derived from the clipped hK2 complex at 50 kDa, since onlythe 10 kDa fragment remains bound to the inhibitor. In summary, sequenceanalysis of the blot shows that about 73% of the hK2 is clipped atAvg.145, 13% is intact hK2, ˜11% is bound to the blocked inhibitor and˜2% is in a complex with ACT.

[0124] Since the blocked inhibitor was present in complex with both theintact (64 kDa) and the clipped fragment of hK2 (50 kDa), it wasnecessary digest the complex with trypsin and sequence the internalpeptide fragments to establish the identity of the inhibitor. Threelanes of purified tissue hK2 identical to FIG. 3 were run and the 50 kDabands excised from the SDS-PAGE and digested with trypsin. The trypticfragments were then separated by the capillary C18 reversed-phase HPLCmicroblotter and automatically blotted onto PVDF. The chromatogramshowing the resolution of the tryptic digest of the 50 kDa band is seenin FIG. 4. Each of the indicated peaks was sequenced. A database searchof the internal sequences revealed that the other protein present at 50kDa was a serine protease inhibitor designated Protease Inhibitor-6,PI6. A total of 24 peptide fragments of PI6 were positively identified,comprising more than 70% of the total PI-6 protein mass. As expected, nopeptide fragments of the PI-6 blocked N-terminus, or the fragments afterthe PI-6 residue 341 reactive site were detected. HK2 fragments wereidentified in peaks 2, 5, 15, 18, and 19. This represents five of the 7possible tryptic fragments of hK2 145-237. No tryptic fragments from thehK2 1-145 region were found, consistent with the sequence informationfor the 50 kDa band in FIG. 3. No unidentified sequences were detected.

Discussion

[0125] These results demonstrate the presence of a novel prostaticcomplex between hK2 and the cytoplasmic serine protease inhibitor, PI-6.This is the first evidence of a tissue-specific inhibitor of hK2 and hasimplications for the regulation of hK2 activity and function in theprostate. It is equally compelling that the inhibitor is identified asPI-6, a relatively newly discovered cytoplasmic inhibitor which has notbeen reported in association with a cancer-related protease.

[0126]FIG. 3 indicates that hK2-PI6 represents approximately 10% of theendogenous hK2. FIG. 1 shows hK2-PI6 to be elevated in tumor over benigntissue.

[0127] The finding of a tissue inhibitor of hK2 is important since hK2has been shown to be highly correlated with prostate cancer. hK2 iselevated in the serum of prostate cancer patients hK2 is elevated in theepithelium of poorly differentiated prostate tissue compared to benigntissues. hK2 has been shown to activate urokinase-type plasminogenactivator, and to inactivate plasminogen activator inhibitor-1. hK2cleaves and inactivates IGFB3, thus providing an enhanced environmentfor metastatic cell growth. hK2 may be involved in the activation ofPSA.

[0128] The reasons for the interaction of hK2 and PI-6 are less clear.hK2 has been shown to be secreted by LNCaP cells and is thereforethought to be an extracellular protease. The reaction between hK2 andPI-6 may therefore result from abnormal intracellular hK2 activation aspart of a defective oncolytic pathway or the leakage of PI-6 fromdamaged or necrotic neoplastic tissue. In either case the presence ofhK2-PI6 may well prove to be a valuable marker for the detection ofprostate cancer.

[0129] It should be noted that hK2-PI6 was not detected in normalseminal plasma (data not shown), indicating that this inhibitor istissue specific and is not part of the normal pathway of PSA and hK2secretion into the glandular lumen of the prostate. It is possible thatseminal levels of hK2-PI6 may become detectable in patients withprostate cancer.

[0130]FIGS. 2 and 3 indicate that 75% of the hK2 in these tissues isclipped at arginine 145. For accurate assessment of the 64 kDa complexin crude extracts analyzed by Western blot it was necessary to run gelsunder non-reducing conditions, as in FIG. 1, to maintain the hK2 intactas a single 33 kDa polypeptide held together by disulfide bonds. Thereactive serine in hK2 which forms the covalent bond with serpins isresidue 189, which is C-terminal to the 145 clip. In FIG. 2, lane 5,PF1D215.1 was shown to recognize the 10 kDa fragment of hK2 146-237, andto detect the 50 kDa PI-6 complex consisting of the hK2 146-237covalently attached to PI-1. PF1D215 was only used on immunoaffinitypurified hK2 since this monoclonal antibody would otherwise cross-reactwith the 20 to 50-fold higher levels of PSA in crude tissue extracts.

[0131] Since clipped hK2 has been shown to be inactive it is likely thatthe hK2-PI6 complex formed prior to cleavage. PSA, which contains asimilarly susceptible site at residue 145, showed negligible cleavage byWestern blot under reducing conditions (data not shown).

[0132] The observation that hK2 is largely clipped has previously beendescribed in seminal plasma and prostate tissue extracts (19). Minorlevels of higher molecular mass forms of hK2 were detected in the tissueextracts but were not identified in those studies.

[0133] The finding of an hK2-PI6 complex is intriguing not only for thepresence of hK2, but the identification of PI-1. PI-6 has homology toother serpins members of the ovalbumin family such as antithrombin III,PAI-1, PAI-2, maspin etc (20; 27). It is present in a number of tissuesincluding human placenta, kidney, heart, skeletal tissue and platelets.PI-6 has been shown to be cytoplasmically localized and so its mostlikely physiological role would be expected to be intracellularprotection against internal proteolytic damage. However, no specificrole or endogenous protease complex has been identified. Unlike PAI-2which can function as an intracellular protease inhibitor but can alsobe secreted as an extracellular inhibitor, PI-6 appears to beexclusively intracellular. Attempts to engineer a promoter to forcesecretion of PI-6 rendered it inactive, possibly due to glycosylation(21).

[0134] PI-6 strongly inhibits trypsin, urokinase and factor 11 and hasalso been shown to have inhibitory activity towards chymotrypsin-likeproteases (28). In the current study the chymotrypsin-like PSA did not,however, show evidence of complex with PI-6 in tissue extracts.

EXAMPLE II Characterization of PI-6 In Prostate Tissues

[0135] A number of questions are raised by this study, which relate toboth hK2 and PI-6. Is the level of PI-6 itself variable in tumorcompared to benign tissue and is this inhibitor correlated withoncogenic development. To answer these questions, PI-6 antibodies weredeveloped and PI-6 expression in tumor versus benign or normal tissueswere compared. This study may be useful in correlating these findings tothe staging and patient outcomes.

Materials and Methods

[0136] Expression and Purification of PI-6

[0137] BL2Ide3 cells transformed with PNID were grown in LB+Kanamycin(30 ug/ml) till OD₆₀₀₌0.7, then induced with 1 mM IPTG for two hours.The cells were harvested by centrifugation at 5K (JA 14) for 10 minutes(NB 9347: 88).

[0138] The cell pellet from 500 ml of E.coli culture was resuspended in20 ml sonicate buffer (300 nM NaCl, 50 mM Sodium Phosphate pH8.0, 2 mMB-Mercaptoethanol) plus protease inhibitor cocktail. The resuspendedcells were sonicated 4 times for 15 seconds. The supernatant and celldebris were separated by centrifugation at 9K (JA 20) for 20 minutes

[0139] The supernatant was incubated with 5 ml prewashed (with sonicatebuffer ) Cobalt resin at 4° C. for 30 minutes. The resin was collectedby centrifugation at 2K (JA 20) for 5 minutes. The resin was then washedwith 20 ml of the wash buffer (300 mM NaCl, 50 mM Sodium Phosphate pH6.0, 10% glycerol, 2 mM 2-Mercaptoethanol) three times, each time shakenat 4° C. for 20 minutes, then collected with centrifugation. PI-6 waseluted twice with the wash buffer plus 400 mM Imidazole. The supernatantwas collected by centrifugation at 2K (JA20) for 5 minutes after shakingat 4° C. for 20 minutes.

[0140] All the fractions from the expression and purification werechecked on SDS gel. It seems the majority of the expressed PI-6 isinsoluble. The soluble fraction was about 90% purified by Cobalt resin.The purified protein was dialyzed against PBS plus 2 mMB-mercaptoethanol. The concentration of the purified PI-6 was measuredby Chemi Imager Analysis based on the density comparison with thelyophilized PI-6. The yield from the 500 ml E.coli culture was about 1mg.

[0141] The pellet after sonication was washed with 1% Triton X-100, 1MNaCl, and 8M Urea sequentially. The insoluble fraction was only solublein 8M Urea.

[0142] Generating antibodies against PI-6

[0143] The α-PI-6 polyclonal antibody (α-PI-6) was generated byinjecting rabbit with the purified PI-6 by using methods that arecommonly known to one skilled in the art.

[0144] hK2 /PI-6 Complex Formation

[0145] 1. Materials used:

[0146] pure hK2: 10 ng/ul

[0147] PI-6: 180 ng/ul purified from E.coli

[0148] 2. Reaction set up:

[0149] 6 ul pure hK2 (30 ng)+14 ul PBS

[0150] 6 ul PI-6(540 ng)+14 ul PBS

[0151] 6 ul PI-6 (540 ng)+6 ul pure hK2 (30 ng)+8 ul PBS

[0152] The reactions were incubated at room temperature for one hour,then 20 ul of loading buffer was added to each of them. The reactionswere then boiled for three minutes and loaded in duplicate of 20 ul eachon the 4-20% SDS gel. Western blotting followed and HK1G568.1 (5 ug/ml)or α-PI-6 (2 ug/ml) were used as primary antibodies. The secondaryantibodies for both sets were diluted 1:5,000.

Results

[0153]FIG. 5 is a Western blot of hK2-PI6 complex formed in vitro withα-PI-6 antibody of the present invention and hK2 antibody. This figureshows that purified PI-6 formed a complex with purified hK2. Thiscomplex was recognized by both α-PI-6 polyclonal and α-hK2 monoclonal(HK1G586.1) antibodies. PI-6 did not complex with the zymogen,enzymatically inactive form of hK2 (data not shown).

[0154]FIG. 6 shows a Western blot of prostate tissue extracts probedwith the α-PI-6. The α-PI-6 antibody was generated by immunization ofrabbits with E.coli-expressed PI-6. Both benign (B) and cancer tissue(C) extracts were obtained from three different tissues. The Westernblot shows that the α-PI-6 antibody recognized not only PI-6, but alsohK2-PI6 complex, and other apparent complexes of PI-6. The Western blotalso shows that the level of hK2-PI6 complex is higher in cancer than inthe benign tissues.

[0155] The large band at the lower portion of the gel is consistent withthe molecular weight of free PI-6. This gel demonstrated that PI-6 ispresent in prostate tissue in significant levels, and that it appears tobe in complex with several other proteins. The fact that the level ofhK2-PI-6 complex is higher in cancer tissue than benign tissue indicatesthat PI-6 may be playing a role in the regulation of some processesassociated with cancer development.

[0156]FIG. 7 shows a Western blot of a prostate tissue extract. Lane 1was probed with HK1G586.1 to detect hK2 forms. In lane 1 the free hK2and the 64 kDa complex is seen, similar to FIG. 1. Lane 2 was probedwith the α-PI-6 antibody. In lane 2 the free PI-6 is detected atapproximately 42 kDa and an equivalent amount of hK2-PI6 complex is seenat exactly the same 64 kDa mobility as seen with the anti-hK2 antibody.

[0157] Again, it is a discovery of the present invention that PI-6 ispresent in prostate tissues. It is also a discovery of the presentinvention that PI-6 may be associated with cancer. The discovery thatPI-6 is complexed with an identified endogenous protease may suggestthat PI-6 could be involved in an oncolytic process, either pro or con.

[0158] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

[0159] The present invention may be embodied in other specific formswithout departing from its essential characteristics. The describedembodiment is to be considered in all respects only as illustrative andnot as restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of the equivalence ofthe claims are to be embraced within their scope.

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What is claimed is:
 1. An isolated and substantially pure complexconsisting of protease inhibitor-6 (PI-6) and human kallikrein 2 (hK2).2. The complex of claim 1 , wherein the complex consists of PI-6 andintact hK2, and the complex has a molecular weight of about 64 kDameasured by non-reducing polyacrylamide gel technique.
 3. The complex ofclaim 1 , wherein the complex consists of PI-6 and a fragment of hK2,and the complex has a molecular weight of about 50 kDa measured byreducing polyacrylamide gel technique.
 4. The complex of claim 3 ,wherein the fragment of hK2 is a clipped hK2 clipped at arginine
 145. 5.The complex of claim 1 being isolated from a prostate tissue.
 6. Thecomplex of claim 1 existing at an elevated level in a prostate tumortissue sample.
 7. An antibody that is specifically immunoreactive with acomplex consisting of protease inhibitor-6 (PI-6) and human kallikrein 2(hK2).
 8. The antibody of claim 7 , wherein the antibody is a polyclonalantibody.
 9. The antibody of claim 7 , wherein the antibody is amonoclonal antibody.
 10. A method of detecting or determining in asample a complex consisting of protease inhibitor-6 (PI-6) and humankallikrein 2 (hK2), the method comprising the steps of: (a) contactingan amount of an agent which specifically binds to the hK2-PI6 complex tobe detected with the sample under a condition that allows the formationof a ternary complex comprising the agent and the hK2-PI6 complex; and(b) detecting or determining the presence or amount of the ternarycomplex.
 11. The method of claim 10 , wherein the agent is an antibody.12. The method of claim 11 , wherein the antibody is a monoclonalantibody.
 13. The method of claim 10 , wherein the agent is an antibodyand wherein the antibody is attached to a solid phase.
 14. The method ofclaim 10 , wherein the agent is an antibody and wherein the antibodycomprises a detectable label or binds to a detectable label to form adetectable complex.
 15. The method of claim 10 , wherein the sample is amammalian tissue sample.
 16. The method of claim 15 , wherein the agentis an antibody.
 17. The method of claim 15 , wherein in step (b), thecomplex is detected by a second agent which comprises a detectable labelor which binds to a detectable label to form a detectable complex. 18.The method of claim 17 , wherein the second agent is an antibody. 19.The method of claim 15 , wherein the mammalian tissue is a humanprostate tissue.
 20. The method of claim 19 , wherein the human prostatetissue is a prostate cancer tissue.
 21. The method of claim 10 , whereinthe sample is a sample of human physiological fluid.
 22. The method ofclaim 21 , wherein the human physiological fluid is selected from thegroup consisting of serum, seminal plasma, urine, and blood.
 23. Animmunoassay for detecting hK2-PI6 complexes in a biological fluid samplecomprising the steps of: (a) providing a first antibody that recognizesone epitope of the hK2-PI6 complex, and a second antibody thatrecognizes another epitope of the hK2-PI6 complex, (b) contacting thefirst antibody with the sample under a condition that allows the firstantibody to bind to the complex to form a first complex that comprisesthe first antibody and the hK2-PI6 complex, (c) contacting the secondantibody with the first complex under a condition that allows the secondantibody to bind to the hK2-PI6 complex to form a second complex thatcomprises the first antibody, the second antibody and the hK2-PI6complex, and (d) detecting and determining the second complex as ameasurement of the hK2 PI6 complex contained in the sample.
 24. Theimmunoassay of claim 23 , wherein the first and second antibodies arerespectively an antibody that is specifically immunoreactive for thehK2-PI6 complex.
 25. The immunoassay of claim 23 , wherein one of thefirst and second antibodies is an antibody against hK2, and another ofthe first and second antibodies is an antibody against PI-6.
 26. Theimmunoassay of claim 23 , wherein one of the first and second antibodiesis bound to a solid phase, and another of the first and secondantibodies is labeled with a detectable label.
 27. A diagnostic kit fordetecting or determining in a sample a complex consisting of PI-6 andhK2, the kit comprising a known amount of an agent which specificallybinds to the complex, wherein the agent is detectably labeled or bindsto a detectable label.
 28. The diagnostic kit of claim 27 , wherein thesample is a sample of human physiological fluid.
 29. The diagnostic kitof claim 27 , wherein the agent comprises an antibody that specificallybinds to the complex.
 30. The diagnostic kit of claim 29 furthercomprising a solid phase capable of having the antibody attachedthereto.
 31. The diagnostic kit of claim 27 , wherein the sample is amammalian tissue sample.
 32. The diagnostic kit of claim 31 , whereinthe mammalian tissue sample is a human prostate tissue sample.
 33. Thediagnostic kit of claim 29 , wherein the antibody is a monoclonalantibody.
 34. A diagnostic kit for detecting or determining in a samplea complex consisting of PI-6 and hK2, the kit comprising a known amountof a first agent which specifically binds to the complex, wherein thefirst agent is detectably labeled or binds to a detectable label, and aknown amount of a second agent which specifically binds to the complex,wherein the second agent is bound to a solid support.
 35. The kit ofclaim 34 , wherein the first agent comprises an antibody that isspecifically immunoreactive with the complex, and the second agentcomprises an antibody that is specifically immunoreactive with thecomplex.
 36. The kit of claim 34 , wherein one of the first and secondagents comprises an antibody that is specifically immunoreactive withhK2, and the other of the first and second agents comprises an antibodythat is specifically immunoreactive with PI-6.
 37. A diagnostic methodfor determining the presence or absence of prostate cancer comprising:(a) contacting an amount of an agent, which specifically binds to acomplex consisting of PI-6 and hK2, with a sample obtained from a humancontaining the complex under a condition sufficient to allow theformation of a ternary complex comprising the agent, the PI-6, and thehK2; and (b) determining the amount of the ternary complex in the sampleand correlating the amount of the ternary complex to the presence orabsence of prostate cancer in the human.
 38. The diagnostic method ofclaim 37 , wherein the sample is a sample of physiological fluid. 39.The diagnostic method of claim 37 , wherein the agent comprises anantibody.
 40. The diagnostic method of claim 37 , wherein the sample isa mammalian tissue sample.
 41. The diagnostic method of claim 40 ,wherein the mammalian tissue sample is a prostate tissue sample.
 42. Adiagnostic method for determining the presence or absence of prostatecancer comprising: (a) contacting an amount of an agent, whichspecifically binds to PI-6, with a sample obtained from a humancontaining the PI-6 under a condition sufficient to allow the formationof a binary complex comprising the agent and the PI-6, wherein thesample is selected from a group consisting of a prostate tissue sample,serum, seminal plasma, urine and blood; and (b) determining the amountof the complex in the sample and correlating the amount of the complexto the presence or absence of prostate cancer in the human.
 43. Thediagnostic method of claim 42 , wherein the sample is a prostate tissuesample.
 44. The diagnostic method of claim 42 , wherein the agentcomprises an antibody.
 45. A method for detecting and determininghK2-PI6 complexes in a tissue specimen, wherein the tissue specimencontains free hk2, free PI-6 and hK2-PI6 complex, the method comprisingthe steps of: (a) providing a first antibody that is specific for thefree hK2, a second antibody that is specific for the free PI-6, and athird antibody that recognizes and binds the hK2-PI6 complex; (b)contacting the first and second antibodies with the tissue specimenunder a condition that all the free hk2 and free PI-6 are blocked by thefirst and the second antibodies respectively; (c) contacting the thirdantibody with the tissue specimen under a condition that allows thethird antibody to bind the hK2-PI6 complex contained in the tissuespecimen to form a ternary complex comprising the antibody and thehK2-PI6 complex; and (d) detecting or determining the presence or amountof the ternary complex.
 46. The method of claim 45 , wherein the thirdantibody is an antibody against hK2, which antibody recognizes and bindsnot only free hK2 but also the hK2-PI6 complex.
 47. The method of claim45 , wherein the third antibody may be an antibody against PI-6, whichantibody recognizes and binds to not only free PI-6, but also thehK2-PI6 complex.